Fig 1.
Ogerin inhibits TGF-β induced myofibroblast differentiation in primary human lung fibroblasts in a dose dependent manner.
A: Primary human lung fibroblasts derived from donors with non-fibrotic or IPF diagnosis co-treated with 1ng/mL TGF-β and/or 50–150μM Ogerin for 72 hours. Whole cell lysates were analyzed for αSMA expression level via Western Blot. αSMA Band expression standardized to β-Tubulin, expressed as percent TGF-β induced. n = 3 per donor per treatment group, dots representative of technical replicates with bars representative of technical replicate range. Statistics omitted for clarity, individual plots with statistical analysis provided in S1 Fig. B: Primary human lung fibroblasts (Non-Fibrotic Donor #1) grown on glass microscope slides co-treated with 1ng/mL TGF-β and/or 100μM Ogerin for 72 hours. Cells fixed, permeabilized and stained for αSMA (Green) and nuclei (DAPI, Blue). Representative confocal images with 50μM scale bar shown in 200x image, and 400x inset. Full dataset provided in S2 Fig.
Fig 2.
Ogerin inhibits TGF-β induced collagen production in primary human lung fibroblasts at the transcriptional level.
A: Primary human lung fibroblasts derived from non-fibrotic or IPF patients were co-treated with 1ng/mL TGF-β and/or 50–150μM Ogerin for 72 hours. Cell culture supernatant was analyzed for secreted Col1A1 via slot blot. Band densitometry was standardized as percent TGF-β induced. n = 3 per donor per treatment group, bars representative of technical replicate range. Statistics omitted for clarity, individual plots with statistical analysis provided in S3B and S3C Fig. Primary human lung fibroblasts derived from non-fibrotic or IPF patients co-treated with 1ng/mL TGF-β and/or 50–150μM Ogerin for 48 hours. Col1A1 and Col3A1 mRNA transcript levels analyzed via qRT-PCR. Transcript abundance standardized to 18s rRNA by the ΔΔCt method and expressed as percent TGF-β induced. Statistics omitted for clarity, individual plots with statistical analysis provided in S4 and S5 Figs.
Fig 3.
Ogerin partially reverses an established, TGF-β induced myofibroblast phenotype in primary human lung fibroblasts.
Primary human lung fibroblasts derived from non-fibrotic or IPF patients were treated +/- 1ng/mL TGF-β (T) or Vehicle (V) for 24 hours. After 24-hour incubation, TGF-β containing media was removed and cells were rinsed with PBS. From 24–72 hours, cells were treated +/- 150μM Ogerin (O) or DMSO Vehicle (V). A: Whole cell lysates were analyzed for αSMA expression level via Western Blot. αSMA band expression was standardized to β-Tubulin, expressed as a percent of TGF-β induced. n = 3 per donor per treatment group, dots representative of technical replicates with SEM. * = p < 0.05 by One Way ANOVA with Tukey’s Post-Hoc Test for Multiple Comparisons. B: Cell culture supernatant was analyzed for secreted Col1A1 via slot blot. Band expression was standardized as a percent of TGF-β induced. n = 3 per donor per treatment group, dots representative of technical replicates with SEM. * = p < 0.05 by One-Way ANOVA with Tukey’s Post-Hoc Test for Multiple Comparisons.
Fig 4.
Prolonged treatment of Ogerin in the presence or absence of TGF-β does not induce apoptosis or negatively impact cellular viability.
A: Primary human lung fibroblasts derived from non-fibrotic or IPF patients were treated +/- DMSO Vehicle, +/- 1ng/mL TGF-β, +/- 50–150μM Ogerin, or 5 μg/mL Puromycin (P) for 72 hours. Whole cell lysates were analyzed for cleaved PARP expression level via Western Blot with GAPDH and β-Tubulin as loading controls, n = 3 per donor per treatment group. B: Primary human lung fibroblasts derived from non-fibrotic or IPF patients were treated with the following reagents: +/- DMSO Vehicle, +/- 1ng/mL TGF-β, +/- 50–150μM Ogerin, or 5 μg/mL Puromycin for 72 hours. Following this incubation period, Alamar Blue cell viability reagent was added and allowed to incubate for 4 hours. Fluorescence intensity (Excitation 535nm, Emission 595nm) measured and expressed as a percent of DMSO Vehicle control. n = 8 per donor per treatment group, bars representative of technical replicate average +/- SEM. Representative images of cells following 72-hour treatment available in S6 Fig.
Fig 5.
Ogerin inhibits TGF-β induced gene transcription in a SMAD reporter assay.
Ogerin does not inhibit TGF-β induced SMAD3 phosphorylation, nuclear localization, or chromatin binding in primary human lung fibroblasts. A: HEK293 cells stably transfected with a TGF-β responsive luciferase element under the transcriptional control of upstream SMAD binding elements co-treated for 24 hours +/- 1-10ng/mL TGF-β and/or +/- 50–150μM Ogerin. Cells were lysed with luciferase substrates and measured for luminescence intensity, expressed as a percent of control. n = 8 per treatment group, bars representative of technical replicate average +/- SEM. B: Primary human lung fibroblasts derived from non-fibrotic and IPF patients were co-treated with 1ng/mL TGF-β and/or 150μM Ogerin for 40 minutes. Whole cell lysates were analyzed for phosphorylated SMAD3 expression via Western Blot. Phosphorylated SMAD3 band intensity was standardized to total SMAD3 band intensity and expressed as a percent of TGF-β induced. n = 3 per donor per treatment group, dots representative of technical replicates with SEM. Cytoplasmic, soluble nuclear, and chromatin bound subcellular fractionations analyzed for total and phosphorylated SMAD3, and for fractionation efficiency utilizing the same samples on a parallel blot. n = 1 per treatment group per fraction. * = p < 0.05 by One-Way ANOVA with Tukey’s Post-Hoc Test for Multiple Comparisons.
Fig 6.
Ogerin induces Gαs signaling as evident by PKA dependent CREB phosphorylation.
A: Primary human lung fibroblasts derived from non-fibrotic or IPF patients were co-treated with 1ng/mL TGF-β and/or 150μM Ogerin for 40 Minutes. Whole cell lysates analyzed for phosphorylated CREB expression via Western Blot. Phosphorylated CREB intensity was standardized to total CREB expressed as a percent of control. n = 3 per donor per treatment group, dots representative of technical replicates with SEM. B: Primary human lung fibroblasts derived from non-fibrotic or IPF patients pre-treated with 20μM of the PKA Inhibitor H-89 for 30 minutes followed by +/- 30μM Forskolin or +/- 150μM Ogerin for 40 Minutes. CREB phosphorylation was analyzed as described for 6A. * = p < 0.05, NS = Not Significant by One -Way ANOVA with Tukey’s Post-Hoc Test for Multiple Comparisons.
Fig 7.
Ogerin’s activation Gαs signaling and inhibition of basal and TGF-β induced collagen gene transcription is potentiated by acidic pH.
A: Primary human lung fibroblasts derived from non-fibrotic or IPF patients were treated +/- 100μM Ogerin for 40 Minutes in pH adjusted PBS. Whole cell lysates were analyzed for phosphorylated CREB expression via Western Blot. Phosphorylated CREB intensity standardized to total CREB expressed as a percent of control. n = 3 per donor per treatment group, dots representative of technical replicates with SEM. B: Primary human lung fibroblasts derived from non-fibrotic subjects or those with IPF were co-treated with 1ng/mL TGF-β and/or 100μM Ogerin for 48 hours in pH adjusted, CO2 Independent media. Col1A1 and Col3A1 mRNA transcript levels were analyzed via qRT-PCR. Transcript abundance was standardized to 18s rRNA by the ΔΔCt method and expressed as percent TGF-β induced. ^ = p < 0.05, NS = Not Significant by Two -Way ANOVA with Multiple Comparisons of cells means regardless of rows and columns. * = p < 0.05, by One-Way ANOVA with Tukey’s Post-Hoc Test for Multiple Comparisons.
Fig 8.
Ogerin inhibits TGF-β induced gene transcription, collagen secretion and myofibroblast differentiation in human dermal, intestinal, and orbital fibroblasts.
A: Human Dermal, Intestinal, and Orbital fibroblasts were co-treated with TGF-β (5ng/mL Dermal, 1ng/mL Intestinal and Orbital) and/or 150μM Ogerin for 48 hours. Col1A1 and ACTA2 mRNA transcript levels were analyzed via qRT-PCR. Transcript abundance standardized to 18s rRNA by the ΔΔCt method and expressed as a fold change of control. B: Human Dermal, Intestinal, and Orbital fibroblasts co-treated with TGF-β (5ng/mL Dermal, 1ng/mL Intestinal and Orbital) and/or 150μM Ogerin for 72 hours. Cell culture supernatant analyzed for secreted Col1A1 via slot blot. Band expression was standardized as percent TGF-β induced. C: Human Dermal, Intestinal, and Orbital fibroblasts co-treated with TGF-β (5ng/mL Dermal, 1ng/mL Intestinal and Orbital) and/or 150μM Ogerin for 72 hours. αSMA Band expression was standardized to β-Tubulin, expressed as percent TGF-β induced. n = 3 per cell line per treatment group, dots representative of technical replicates with SEM. * = p < 0.05, by One -Way ANOVA with Tukey’s Post-Hoc Test for Multiple Comparisons.
Fig 9.
Ogerin does not inhibit TGF-β induced SMAD3 phosphorylation, and induces Gαs signaling in human dermal, intestinal, and orbital fibroblasts.
A,B: Human Dermal, Intestinal, and Orbital fibroblasts were co-treated with TGF-β (5ng/mL Dermal, 1ng/mL Intestinal and Orbital) and/or 150μM Ogerin for 40 minutes. Whole cell lysates were analyzed for phosphorylated SMAD3 expression via Western Blot. Phosphorylated SMAD3 band intensity was standardized to total SMAD3 band intensity and expressed as a percent of TGF-β induced. Whole cell lysates were analyzed for phosphorylated CREB expression via Western Blot. Phosphorylated CREB intensity was standardized to total CREB expressed as a percent of control. * = p < 0.05, by One -Way ANOVA with Tukey’s Post-Hoc Test for Multiple Comparisons.